Bt is a gram-positive bacilliform bacterium which produces a crystal protein at a sporulation stage in the end of a logarithmic growth phase. When an insect orally takes the crystal protein into a gastrointestinal tract, the crystal protein will be subjected to alkaline and enzymatic degradations in a digestive juice so as to show an insecticidal activity which causes an intestinal and systemic paresis. The crystal protein is therefore referred to as ".delta.-endotoxin" (Heimpel, A. M.; Ann. Rev., Entomology 12, 287-322, 1967).
The crystal protein produced by Bt is generally of a form such as a diamond-shaped, bipyramidal and rhomboidal one. The crystal protein is formed with an endospore in a sporangium and released with it from the sporangium (Hannay, C. L.; Nature 172, 1004, 1953).
Bt has been classified on the basis of H-antigen according to the proposal by De Barjac and Bonefoi (Entomophaga 7, 5-31, 1962), and reported to have 24 subspecies (33 strains) including serotypes 1 to 23 and wuhanensis which has no H-antigen (Toshihiko Iizuka, Chemistry and Biology 27, 287-302, 1989). However, it was found that some different kinds of strains were present in the same serotype (Krywienczyk, J., et al; J. Invertebr. Pathol.; 31, 372-375, 1978, Iizuka, T., et al; J. Sericult. Sci. Japan; 50, 120-133, 1981).
The crystal protein produced by Bt is generally known to selectively show the insecticidal activity to the lepidopterous insects. Recently, subspecies have been found, which produce an irregular and cuboidal crystal protein beside a bipyramidal one (Yoshio Akiba; Jpn. J. Appl. Entomol. Zool., 30, 99-105, 1986, Ohba, M., et al; J. Invertebr. Pathol. 38, 307-309, 1981). Among them, there were reported strains which can show a strong insecticidal activity against a leaf beetle or a larva of a mosquito (Hall, I. M., et al; Mosquito news 37, No.2, 1977).
A crystal protein gene (CP gene) of Bt is usually encoded by a plural of somatic plasmid DNAs. The CP gene has been already cloned and its nucleotide sequence has been also determined in some subspecies (Toshihiko Iizuka, Chemistry and Biology 27, 287-302, 1989).
Further, many researches have been intensively conducted on introduction of such cloned CP genes into a plant body and some of them were reported to succeed, for example, in the case of tobacco (Vaeck, M., et al; Nature 328, 33-37, 1987). The CP gene coding for a crystal protein which shows a stronger insecticidal activity is now sincerely desired.
The lepidopterous insect pests having taken the crystal toxin produced by Bt will stop their feeding behavior several hours later and never do any harm to plants. Almost species of the lepidopterous insect pests will die of intoxication by the crystal toxin about 24 to 72 hours later. The intoxication will be sometimes accompanied by sepsis induced by the presence of spores. Thus, a main reason of the death is due to the crystal toxin which will function only after its dissolution in a bowel of the larva.
Due to such functional mechanism of the crystal toxic protein as well as the fact that the crystal toxin is an easily-degradable polypeptide, it is known that the crystal toxin does no damage to human, pet animals, birds, fishes and plants.
Accordingly, Bt or the crystal toxic protein produced thereby is very potential as a microbial pesticide which does not pollute an environment (BT agent), especially as an insecticide against the lepidopterous insect pests. One or more strains of Bt have been actually used for a long time as the insecticide in agriculture.
The strain of Bt which is most generally used as a commercial product is Bt var. kurstaki HD-1, which will be referred to hereinafter as "HD-1."